2.1 CDK2
Human cyclin-dependent kinase 2 or cell division kinase (CDK2; GenBank Accession No. X61622; Elledge and Spottswood, 1991, EMBO J. 10: 2653-2659; Ninomiya-Tsuji et al., 1991, Proc. Natl. Acad. Sci. USA 88: 9006-9010) is a serine-threonine protein kinase of 298 amino acids that has approximately 65% amino acid identity to a second critical cell cycle regulator, p34cdc2, more commonly known as CDC2. CDK2 is expressed late in G1 or early in S phase, slightly before CDC2, and is pivotal for G1/S transition. CDK2 cannot complement yeast CDC2/CDC28 mutations under all the conditions that CDC2 can, indicating that the two kinases regulate the cell cycle at distinct stages.
CDK2 activity is dependent upon phosphorylation of threonine 160 by CDK-activating kinase (CAK), which occurs when CDK2 complexes with cyclins A and E. Conversely, CDK2 kinase activity is inactivated by dephosphorylation by human KAP (CDK-Associated Phosphatase; Poon and Hunter, 1995, Science 270: 90-93). In particular, competition between KAP and cyclin A determines the phosphorylation state of CDK2. CDK2 phosphorylates pRb, p53, transcription factor E2F, histone H1, and other proteins central to cell cycle control (Higashi et al., 1996, Eur. J. Biochem. 237: 460-467). Other proteins, including p21.sup.cip, and p.sub.27.sup.kip, complex with CDK2 to block its interaction with downstream substrates, as well as blocking CDK2 phosphorylation itself (Adams et al., 1996, Mol. Cell. Biol. 16: 6623-6633). The complex interplay of phase-specific cyclin expression, phosphorylation/dephosphorylation cascades, and other CDK2 interacting proteins such as p21.sup.waf and p27.sup.kip, ultimately plays out through CDK2 activity to determine cell cycle progression.
Deregulation of CDK2 is strongly implicated in mechanisms of carcinogenesis and in the treatment of cancer. DNA tumor viruses transform cells by directly inhibiting Rb tumor-suppressing function (e.g., papilloma viruses and cervical cancer). The Rb then releases negative control of E2F. Normally, the inhibition of Rb is accomplished by phosphorylation of Rb by CDK2 (Nevins, 1992, Science 258: 424-429). CDK2 is implicated in the differentiation of glioma cells (Kokunai et al., 1997, J. Neuro-oncol. 32: 125-133). In human breast carcinoma cells, the anti-cancer agent flavopiridol induces G1 arrest by inhibition of both CDK2 and CDK4 (Carlson et al., 1996, Cancer Res. 56: 2973-2978). Anti-estrogens up-regulate CDK2 inhibitors p21.sup.cip and p27.sup.kip, thus causing reduction in pRb phosphorylation, and decreased cell progression into S phase (Watts et al., 1995, Mol. Endocrinol. 9: 1804-1813). Serum-deprivation of vascular smooth muscle cells is associated with CDK2/p27.sup.kip complex formation, leading to inhibition of CDK2 enzymatic activity (Chen et al., 1997, J. Clin. Invest. 99: 2334-2341). Thus, along with regulation of cyclin A expression, CDK2 activity is the mechanism through which p27.sup.kip acts to inhibit intimal hyperplasia during atherosclerosis and re-stenosis.
To review, CDK2 is implicated in the control of cell cycle progression, transcriptional regulation via E2F, control of cellular differentiation, intracellular signal transduction involving phosphorylation, mechanisms of tumorigenesis, tumor progression and spread, and atherosclerosis and re-stenosis via effects on intimal proliferation. cl 2.2 Cyclin H
Human cyclin H (GenBank Accession No. U11791; Makela et al., 1994, Nature 371: 254-257) is a 323 amino acid protein that complexes with CDK7 to form CDK-activating kinase (CAK; Fisher and Morgan, 1994, Cell 78: 713-724). CAK, in turn, phosphorylates CDK2, as well as various cyclin complexes of CDK2 and CDK4. Thus, like CDK2, cyclin H is centrally implicated in control of cell cycle progression. CAK is also associated with the mammalian transcription factor IIH (TFIIH), a multisubunit complex that is required for transcription and DNA nucleotide excision repair (Drapkin et al., 1996, Proc. Natl. Acad. Sci. USA 93: 6488-6493). Therefore, the role of cyclin H extends beyond cell cycle control to include coordination of the cell cycle with transcription and DNA repair. Dysfunction of TFIIH is implicated in various genetic disorders including xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy (Seroz et al., 1995, Curr. Opin. Genet. Dev. 5: 217-222). In summary, cyclin H is implicated in the control of cell cycle progression, transcriptional control via TFIIH, DNA repair, and various genetic disorders associated with impaired DNA repair.